High molecular weight extracts of Convolvulus arvensis field (field bindweed)

ABSTRACT

A purified bindweed extract is used to inhibit the growth of tumor cells, inhibit the growth of blood vessels, and enhance immune function. The bindweed extract is prepared by removing toxic low molecular weight components of Convolvulus arvensis.

FIELD OF THE INVENTION

This invention relates to obtaining purified bindweed extracts bycollecting high molecular weight components of a homogenized aqueoussolution of bindweed material. These high molecular weight extracts haveunexpectedly been shown to inhibit the growth of two different types ofvirulent tumors in mice. The extracts possess immunopotentiating effectsas evidenced by tumor infiltration by white blood cells in the tumors oftreated animals, and induction of lymphocyte proliferation. They furtherpossess antiangiogenic properties as demonstrated in the chickchorioallantoic membrane assay. The method of extraction allows for theremoval of small molecules, including alkaloids, from the plantmaterial, which are known to be toxic. The bindweed extracts haveutility as low-toxicity, anti-cancer drugs for human and animal use.

BACKGROUND OF THE INVENTION

Recently the concept of using biological response modifiers (BRMs) hasbeen considered for the treatment of cancer. Some BRMs are not directlycytotoxic to tumor cells, but possess qualities which change theenvironment of the organism. Immune stimulation and inhibition of newblood vessel growth are two biological response modification strategieswith potential in the treatment of cancer. Examples of immunestimulators with known anti-tumor activity are Polysaccharide K, beta1,3, glucan, and the Maruyama vaccine. All of which containhigh-molecular weight polysaccharides and/or protein. Examples ofangiogenesis inhibiting molecules include TMP-470, and angiostatin,which have demonstrated anti-tumor activity. While investigatingextracts of field bindweed for anti-tumor activity, we initially triedto isolate low-molecular weight alkaloids, which are known to be toxic,and which we suspected of having traditional chemotherapeutic, ortumor-cytotoxic activity.

It was found that the low-molecular weight extracts, containing thetoxic alkaloids, exhibited little anti-tumor activity, while highmolecular weight extracts, which excluded the toxic alkaloids containedsignificant anti-tumor activity by acting as biological responsemodifiers.

SUMMARY OF THE INVENTION

One object of the invention is to provide a pharmaceutical consisting ofhigh molecular weight extracts of field bindweed (Convolvulus arvensis)which have low toxicity to normal cells and induce anti-tumor effects inanimals, inhibit the growth of blood vessels, and enhance immunefunction in mammals. Preferably these high molecular weight extractshave components less than about 500 Daltons removed. More preferably theremoved components are less than about 1,000. Even more preferably, theremoved components are less than about 3,000 Daltons. Even morepreferably, the removed components are less than about 5000, morepreferably 10,000.

One embodiment of the invention is to isolate the high molecular weightextracts of field bindweed (Convolvulus arvensis) using a molecularweight filter or alternatively precipitation with ammonium sulfate.

A further object of the present invention is to provide a method forisolating high molecular weight extracts of field bindweed (Convolvulusarvensis), which have low toxicity by virtue of removal of toxiclow-molecular weight components of a crude extract. Preferably thecomponents less than about 3000 Daltons are removed. More preferably thecomponents less than about 6,500 Daltons are removed. Even morepreferably, the components less than about 10,000 Daltons are removed.

One embodiment of the invention is to remove the low molecular weightextracts of field bindweed (Convolvulus arvensis) using a molecularweight filter or alternatively precipitation with ammonium sulfate.

A further object of the present invention is to provide a method for useof high molecular weight extracts of field bindweed (Convolvulusarvensis), which have low toxicity to normal cells and induce anti-tumoreffects in animals.

A further object of the present invention is to provide a method for useof high molecular weight extracts of field bindweed (Convolvulusarvensis), which inhibit new blood vessel growth.

A further object of the present invention is to provide a method for useof high molecular weight extracts of field bindweed (Convolvulusarvensis), which exhibits immunoenhancing effects in animals.

These and other objects of the invention will become more apparent byreference to the materials and methods hereinafter set forth.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

We discovered a high molecular weight extract of bindweed thatsurprisingly has low toxicity to normal cells and induces anti-tumoreffects, inhibits the growth of blood vessels, and enhances immunefunction in mammals. We also found that the low molecular weightcomponents of bindweed extract contained alkyloids on the order of300-500 Daltons that were toxic to both normal cells and tumor cells. Inorder to remove the toxic low molecular weight components we used amolecular weight sieve. In general, the method of the invention forobtaining high molecular weight extracts of Convolvulus arvensisconsists of boiling fresh or aged bindweed in an aqueous solution toobtain a brown tea-like mixture. This mixture is then centrifuged orfiltered to remove the solid material to create a solution. It is onlynecessary to remove the 300-500 molecular weight components to removethe toxicity to normal cells. However, we found that the low molecularweight components might nonspecifically stick to the membrane and comeback off. Therefore, preferably, a larger molecular weight sieve isused--on the order of 1000 Daltons, more preferably, 3000 Daltons, evenmore preferably 5000 Daltons. We find that even when molecular weightcomponents of up to 10,000 Daltons are removed, the extract retains itsanti-tumor effects in animals, its ability to inhibit the growth ofblood vessels, and its ability to enhance immune function in mammals. Inthe Examples below, after boiling the fresh or aged bindweed in anaqueous solution to obtain a brown tea-like mixture, the solution isthen passed through a molecular weight filtration device to obtain ahigh molecular weight retentate (BWR), or precipitated using ammoniumsulfate to isolate a high molecular weight precipitate (BWP).Thereafter, the high molecular weight extract is lyophilized, orotherwise concentrated. The extract is subsequently assayed foranti-tumor, immunoenhancing, and anti-angiogenesis activity.

SDS-Page, IEF, and a protein assay, are then used in order to furthercharacterize the components of the extract. The extract is also assayedfor molecular weight using a Superose 12-HR sizing column (PharmaciaBiotech) using known molecular weight standards.

Further features and advantages of the present invention will becomeapparent to those of skill in the art in view of the detaileddescription of the preferred embodiments which follows when consideredtogether with the attached drawings and claims.

Although other material and methods similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described.Example 1 describes the steps required to prepare the high molecularweight extracts of bindweed.

EXAMPLE 1 Preparation of High Molecular Weight Extracts of Bindweed

We have isolated high molecular weight extracts of Convolvulus arvensisas follows. Fresh Convolvulus arvensis was harvested and the seeds, andflowers, were discarded. These portions of the plants were homogenizedusing a Waring blender. To the homogenized plant material, 3 volumes ofdeionized water were added to create a mixture. This mixture was thenboiled for 30 minutes to obtain a tea. The solids were removed from thetea by filtration, and the supernatant retained. The supernatant wascooled and from it two high molecular weight isolates were prepared.Some of the tea supernatant was processed in a CH2 concentrator equippedwith a 3,000 or greater Dalton cartridge. The retentate of theconcentrator (containing only molecules larger than 3,000 Daltons) wasretained, and lyophilized, and is herein referred to as BWR.Alternatively, some of the tea supernatant was combined with ammoniumsulfate to yield a final saturation of 50% ammonium sulfate. Thisresulted in precipitation of some of the components of the teasupernatant. The precipitated components were then collected aftercentrifugation at 25,000 g for 30 minutes, and resuspended in phosphatebuffered saline to form a new solution. This new solution was thenpassed through a ultrafiltration device using a 3,000 Dalton membrane.The retentate was collected, and lyophilized, and herein referred to asBWP. BWP, and BWR, were stored at 4° C. in a dessicator prior to use incharacterization, and anti-tumor, anti-angiogenesis, and immuneparameter studies.

In Examples 2 and 3, the high molecular weight extracts werecharacterized and tested for toxicity.

EXAMPLE 2 Characterization of High Molecular Weight Extracts of Bindweed

SDS-Page analysis of BWR and BWP both showed a generalized smearingpattern, which is a polysaccharide characteristic, with occasionalprotein bands at various molecular weights ranging from greater than200,000 Daltons to 6,500 Daltons. Both extracts were positive for theMolish reaction, which indicates the presence of sugar moieties. Andboth were strongly positive for protein using the bicinchroninic acidassay. The above leading to the conclusion that BWP, and BWR containboth protein and saccharide. FPLC chromatography of BWR, and BWP using aSuperose 12-HR sizing column demonstrate only high molecular weightpeaks with no detection of molecules smaller than 10,000 Daltons.

EXAMPLE 3 Characterization of Toxicity of High Molecular Weight Extractsof Bindweed

Standard chemotherapy screening for cytotoxic anti-tumor agents selectsfor agents with cytotoxic activity at a concentration of 5 mcg/mL. Bothextracts of bindweed were only toxic to cultured tumor cells atconcentrations of 1000 mcg/mL or greater, demonstrating low toxicity.

In Examples 4 through 10 the effects of the high molecular weightextracts, BWR and BWP were analyzed as to anti-angiogenesis activity,anti-tumor activity and effect on immune function.

EXAMPLE 4 Anti-angiogenesis Activity of BWR

The high molecular weight saccharide/protein containing extracts ofbindweed were prepared in accordance with the above-mentioned methods.

The anti-angiogenesis activity of BWR was examined. Angiogenesis wasinduced on chicken egg chorioallantoic membranes by placing a 2 mmmethylcellulose disc containing 10 mcg of heparin onto 11 day oldchicken egg chorioallantoic membranes. Doses of 200, 100, and 50 mcg ofBWR, were concomitantly added to the heparin-containing disc in threesets of 6 eggs each. 6 eggs served as controls. After 4 days, a largewindow was created over the chorioallantoic membranes, and the numbersof new blood vessels on each egg were counted.

A percent inhibition on angiogenesis was obtained according to thefollowing equation:

    Angiogenesis inhibition (%)=(1-Average number of new blood vessels of test group/Average number of new blood vessels of control group)×100

The results obtained are shown in Table 1 below. It is seen that theangiogenesis, or the induction of new blood vessels was significantlyinhibited by the administration of BWR, in a dose-dependent manner.

                  TABLE 1                                                         ______________________________________                                        Angiogenesis inhibition                                                       200 mcg/egg    100 mcg/egg                                                                             50 mcg/egg                                           ______________________________________                                        73%            55%       18%                                                  ______________________________________                                    

EXAMPLE 5 Antitumor Activity of BWR

The antitumor effects of BWR, prepared as described above, wereexamined. Ten 6-week-old mixed-gender Kun Ming mice per group were usedas test animals. S-180 fibrosarcoma cells were subcutaneouslytransplanted to the left inguinal region. Daily for 14 days, BWR wasinjected subcutaneously in the right inguinal region. On the 15th dayafter the transplantation, the tumor was excised and weighed. A percentinhibition on tumor growth was obtained according to the followingequation:

    Tumor growth inhibition (%)=(1-Average tumor weight of test group/Average tumor weight of control group)×100

The results obtained are shown in Table 2 below. It is seen that tumorgrowth was significantly inhibited by the administration of BWR.

                  TABLE 2                                                         ______________________________________                                                Dose         Average    Tumor Growth                                  Group   Dose In mg   Tumor Weight                                                                             Inhibition (%)                                ______________________________________                                        Control 0            1.4                                                      BWR     1 × 14 0.32       77                                            ______________________________________                                    

EXAMPLE 6 Antitumor Effects of BWP

Additionally, the antitumor effects of BWP were examined. Ten 6-week-oldmixed-gender Kun Ming mice per group were used as test animals. S-180cells were subcutaneously transplanted to the left inguinal region.Daily for 14 days, BWP was injected subcutaneously in the right inguinalregion. On the 15th day after the transplantation, the tumor was excisedand weighed. A percent inhibition on tumor growth was obtained accordingto the following equation:

    Tumor growth inhibition (%)=(1-Average tumor weight group/Average tumor weight control group)×100

The results obtained are shown in Table 3 below. It is seen that tumorgrowth was significantly inhibited by the administration of BWP.

                  TABLE 3                                                         ______________________________________                                                Dose         Average    Tumor Growth                                  Group   Dose In mg   Tumor Weight                                                                             Inhibition (%)                                ______________________________________                                        Control 0            2.5                                                      BWP     1 × 14 0.6        74                                            ______________________________________                                    

EXAMPLE 7 Further Antitumor Effects of BWP

The antitumor effects of BWP were examined further. Ten 6-week-old C57mixed-gender mice per group were used as test animals. LLC, Lewis LungCarcinoma Cells were subcutaneously transplanted to the left inguinalregion. Daily for 21 days, BWR was injected subcutaneously in the rightinguinal region. On the 22^(nd) day after the transplantation, the tumorwas excised and weighed. A percent inhibition on tumor growth wasobtained according to the following equation:

    Tumor growth inhibition (%)=(1-Average tumor weight group/Average tumor weight control group)×100

The results obtained are shown in Table 4 below. It is seen that tumorgrowth was significantly inhibited by the administration of BWP.

                  TABLE 4                                                         ______________________________________                                                Dose         Average    Tumor Growth                                  Group   Dose In mg   Tumor Weight                                                                             Inhibition (%)                                ______________________________________                                        Control 0            3.5                                                      BWP     1 × 21 1.33       62                                            ______________________________________                                    

EXAMPLE 8 Further Antitumor Effects of BWR

The antitumor effects of BWR were examined further. Ten 6-week-oldmixed-gender Kun Ming mice per group were used as test animals. S-180cells were subcutaneously transplanted to the left inguinal region.Daily for 14 days, BWP was injected intraperitoneally. On the 15^(th)day after the transplantation, the tumor was excised and weighed. Apercent inhibition on tumor growth was obtained according to thefollowing equation:

    Tumor growth inhibition (%)=(1-Average tumor weight group/Average tumor weight control group)×100

The results obtained are shown in Table 5 below. It is seen that tumorgrowth was significantly inhibited by the administration of BWR.

                  TABLE 5                                                         ______________________________________                                                Dose         Average    Tumor Growth                                  Group   Dose In mg   Tumor Weight                                                                             Inhibition (%)                                ______________________________________                                        Control 0            2.5                                                      BWR     1 × 14 0.8        68                                            ______________________________________                                    

The tumors from this study were embedded in paraffin, stained, andexamined microscopically. It was found that the excised tumors from thetreated group contained large numbers of lymphocytes and monocytes, andonly 10% tumor tissue. The weight of tumor tissue from the tumor tissuein the treated groups was therefore only 10% of that recorded, or 0.08grams rather than 0.8 grams. Therefore the actual inhibition was 96.8%.These results also demonstrate that BWR contains a quality whichenhances the immune response to tumor tissue.

EXAMPLE 9 Effects of BWR and BWP on Human Lymphocyte Growth

The effects of BWR, and BWP on human lymphocyte growth in culture wereexamined. Human lymphocytes were harvested using venipuncture, andsubsequently isolated by use of a centrifuged density gradient. Theywere then incubated in a commercial lymphocyte culture medium (AIM V,containing interleukin 2 and 2 mercaptoethanol) in an atmospherecontaining 95% air, 5% carbon dioxide, at 37° C., for 3 days. Thelymphocytes were then counted using a Coulter Epics XL flow cytometer. Apercent increase in lymphocyte growth was obtained according to thefollowing equation:

    Lymphocyte growth increase (%)=(Average number of lymphocytes of test group/Average number of lymphocytes of control group×100)-100

The results obtained are shown in Table 6 below. It is seen that BWR,and BWP induced lymphocyte proliferation, in a dose-dependent manner.

                  TABLE 6                                                         ______________________________________                                        Dose     0         .8     4       20  100                                     (mcg/ml)                                                                      BWR      0         0      20      0   35                                      BWP      0         12     35      20  46                                      ______________________________________                                    

EXAMPLE 10 Effects of BWR and BWP on Human Phagocytic Activity

The effects of BWR, and BWP on human phagocyte activity were examined.Two buffy-coat samples were prepared by centrifuging tubes containinganti-coagulated human blood from two subjects. The samples were thendivided in two. To one buffy-coat from each subject, 2 micrograms ofBWR, and BWP were added. One buffy-coat from each subject served ascontrol. All samples were incubated for 5 hours. Then 30 milligrams offreshly rehydrated baker's yeast was added to all samples. After onehour, a stock 2× solution of acridine orange stain was added to eachsample. An aliquot of each sample was then placed on a microscope slide.The percentage of phagocytes containing intracellular baker's yeast fromeach sample was recorded.

The results revealed an average increase of 85% in the percentage ofphagocytes containing intracellular baker's yeast in the treated samplescompared to the controls. This elucidates another mechanism by which BWRand BWP stimulate the immune system.

What is claimed is:
 1. A pharmaceutical composition for treating cancer,inhibiting the growth of new blood vessels, and/or enhancing immunefunction in a mammal, said composition comprising: an effective amountof an aqueous extract of Convolvulus arvensis prepared by a processcomprising:homogenizing Convolvulus arvensis plant parts; preparing saidaqueous extract from said plant parts; and substantially removingcomponents from said extract of molecular weight less than about 500Daltons, said pharmaceutical composition further comprising apharmaceutically acceptable vehicle.
 2. The pharmaceutical compositionof claim 1 wherein components of molecular weight less than about 1000Daltons have been removed.
 3. The pharmaceutical composition of claim 2wherein components of molecular weight less than about 3000 Daltons havebeen removed.
 4. The pharmaceutical composition of claim 3 whereincomponents of molecular weight less than about 5000 Daltons have beenremoved.
 5. The pharmaceutical composition of claim 4 wherein componentsof molecular weight less than about 10,000 Daltons have been removed. 6.A method for preparing non-toxic extracts of Convolvulus arvensisconsisting of; removing components with a molecular weight less thanabout 500 Daltons.
 7. The method of claim 6 wherein components with amolecular weight less than about 1000 Daltons are removed.
 8. The methodof claim 7 wherein components with a molecular weight less than about3000 Daltons are removed.
 9. The method of claim 8 wherein componentswith a molecular weight less than about 5000 Daltons are removed. 10.The method of claim 9 wherein components with a molecular weight lessthan about 10,000 Daltons are removed.
 11. The method of claim 6 whereinsaid components are removed using a molecular weight filter.
 12. Themethod of claim 6 wherein said components are removed by precipitationwith ammonium sulfate.
 13. A method for the treatment of cancer in amammal comprising the steps of:administering the pharmaceuticalcomposition of claim 1 in an amount effective to slow or stop the growthof said cancer.
 14. A method for inhibiting blood vessel growth in amammal comprising the steps of:administering the pharmaceuticalcomposition of claim 1 in an amount effective to slow or stop the growthof said blood vessels.
 15. A method for enhancing immune function in amammal comprising the steps of:administering the pharmaceuticalcomposition of claim 1 in an amount effective to enhance said immunefunction.
 16. The method of claim 15 wherein the immune function isselected from the group consisting of lymphocyte growth and phagocyteactivity.
 17. The composition of claim 1, wherein the plant partsexclude seeds and flowers.
 18. The composition of claim 1, wherein theextract is prepared using three volumes of water per volume ofhomogenized plant parts.
 19. The composition of claim 1, wherein solidsare removed from the extract.
 20. The composition of claim 1, whereinthe extract is lyophilized after removal of said components.
 21. Thecomposition of claim 1, wherein the extract is subjected to ammoniumsulfate precipitation.
 22. The composition of claim 21, wherein theextract subjected to precipitation is centrifuged to collect theprecipitates.
 23. The composition of claim 22, wherein the precipitatesare resuspended in phosphate buffer.
 24. The composition of claim 23,wherein the resuspended precipitates are subjected to ultrafiltration toremove said components.